PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry

A number of different approaches have been described to identify proteins from tandem mass spectrometry (MS/MS) data. The most common approaches rely on the available databases to match experimental MS/MS data. These methods suffer from several drawbacks and cannot be used for the identification of proteins from unknown genomes. In this communication, we describe a new de novo sequencing software package, PEAKS, to extract amino acid sequence information without the use of databases. PEAKS uses a new model and a new algorithm to efficiently compute the best peptide sequences whose fragment ions can best interpret the peaks in the MS/MS spectrum. The output of the software gives amino acid sequences with confidence scores for the entire sequences, as well as an additional novel positional scoring scheme for portions of the sequences. The performance of PEAKS is compared with Lutefisk, a well-known de novo sequencing software, using quadrupole-time-of-flight (Q-TOF) data obtained for several tryptic peptides from standard proteins.     

Protein identification methods in proteomics

A combination of high-resolution two-dimensional (2-D) polyacrylamide gel electrophoresis, highly sensitive biological mass spectrometry, and the rapidly growing protein and DNA databases has paved the way for high-throughput proteomics. This review concentrates on protein identification. We first discuss the use of protein electroblotting and Edman sequencing as tools for de novo sequencing and protein identification. In the second part, we highlight matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as one of the main contemporary analytical methods for linking gel-separated proteins to entries in sequence databases. In this context we describe the two main MALDI-MS-based identification methods: (i) peptide mass fingerprinting, and (ii) post-source decay (PSD) analysis. In the last part, we briefly emphasize the importance of sample preparation for obtaining highly sensitive and high-quality MALDI-MS spectra.     

Automated nanoflow liquid chromatography/tandem mass spectrometric identification of proteins from Shewanella putrefaciens separated by two-dimensional polyacrylamide gel electrophoresis

The implementation of nanoflow liquid chromatography offers unique opportunities for automation of proteomics research. We demonstrate that automated nanoflow LC/MS/MS allowed the unambiguous identification of proteins from the omnipotent bacterium Shewanella putrefaciens, based on similarity searches against the completely determined genome of related microorganisms and against non-redundant databases. Total protein extracts were separated by 2-dimensional polyacrylamide electrophoresis. Only 1/20th of a tryptic digest mixture obtained from a single Coomassie Blue stained spot was loaded on the nanoflow LC column using a preconcentration/desalting step, and analyzed on-line on a hybrid quadrupole time-of-flight mass spectrometer with an automated MS-to-MS/MS switching protocol. This method allowed the de novo peptide sequence determination of several tryptic fragments and the identification of different proteins. CopyrightCopyright 2000 John Wiley & Sons, Ltd.     

De novo analysis of protein N-terminal sequence utilizing MALDI signal enhancing derivatization with Br signature

De novo analysis of protein N-terminal sequence is important for identification of N-terminal proteolytic processing such as N-terminal methionine or signal peptide removal, or for the genome annotation of uncharacterized proteins. We introduce a de novo sequencing method of protein N terminus utilizing matrix-assisted laser desorption/ionization (MALDI) signal enhancing picolinamidination with bromine isotopic tag incorporated to the N terminus. The doublet signature of bromine in the tandem mass (MS/MS) spectrum distinguished N-terminal ion series from C-terminal ion series, facilitating de novo N-terminal sequencing of protein. The dual advantage of MALDI signal enhancement by the basic picolinamidine and b-ion selection aided by Br signature is demonstrated using a variety of peptides. The N-terminal sequences of myoglobin and hemoglobin as model proteins were determined by incorporating the Br tag to the N terminus of the proteins and obtaining a series of b-ions with Br signature by MS/MS analysis after chymotryptic digestion of the tagged proteins. The N-terminal peptide was selected for MS/MS analysis from the chymotryptic digest based on the Br signature in the mass spectrum. Identification of phosphorylation site as well as N-terminal sequencing of a phosphopeptide was straightforward.     

Proteome analysis of a human heptocellular carcinoma cell line, HCC-M: an update.

Recently, we reported the proteome analysis of a human hepatocellular carcinoma cell line, HCC-M (Electrophoresis 2000, 21, 1787-1813), using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). From a total of 408 unique spots excised from the 2-DE gel, 301 spots yielded good MALDI spectra. Out of these, 272 spots had matches returned from the database search leading to the identification of these proteins. Here, we report the results on the identification of the remaining 29 spots using nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). First, "peptide tag sequencing" was performed to obtain partial amino acid sequences of the peptides to search the SWISS-PROTand NCBI nonredundant protein databases. Spots that were still not able to find any matches from the databases were subjected to de novo peptide sequencing. The tryptic peptide sequences were used to search for homologues in the protein and nucleotide databases with the NCBI Basic Local Alignment Search Tool (BLAST), which was essential for the characterization of novel or post-translationally modified proteins. Using this approach, all the 29 spots were unambiguously identified. Among them, phosphotyrosyl phosphatase activator (PTPA), RNA-binding protein regulatory subunit, replication protein A 32 kDa subunit (RP-A) and N-acetylneuraminic acid phosphate synthase were reported to be cancer-related proteins.     

Proteomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database

The proteomic definition of plasma membrane proteins is an important initial step in searching for novel tumor marker proteins expressed during the different stages of cancer progression. However, due to the charge heterogeneity and poor solubility of membrane-associated proteins this subsection of the cell's proteome is often refractory to two-dimensional electrophoresis (2-DE), the current paradigm technology for studying protein expression profiles. Here, we describe a non-2-DE method for identifying membrane proteins. Proteins from an enriched membrane preparation of the human colorectal carcinoma cell line LIM1215 were initially fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 4-20%). The unstained gel was cut into 16 x 3 mm slices, and peptide mixtures resulting from in-gel tryptic digestion of each slice were individually subjected to capillary-column reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS). Interrogation of genomic databases with the resulting collision-induced dissociation (CID) generated peptide ion fragment data was used to identify the proteins in each gel slice. Over 284 proteins (including 92 membrane proteins) were identified, including many integral membrane proteins not previously identified by 2-DE, many proteins seen at the genomic level only, as well as several proteins identified by expressed sequence tags (ESTs) only. Additionally, a number of peptides, identified by de novo MS sequence analysis, have not been described in the databases. Further, a "targeted" ion approach was used to unambiguously identify known low-abundance plasma membrane proteins, using the membrane-associated A33 antigen, a gastrointestinal-specific epithelial cell protein, as an example. Following localization of the A33 antigen in the gel by immunoblotting, ions corresponding to the theoretical A33 antigen tryptic peptide masses were selected using an "inclusion" mass list for automated sequence analysis. Six peptides corresponding to the A33 antigen, present at levels well below those accessible using the standard automated "nontargeted" approach, were identified. The membrane protein database may be accessed via the World Wide Web (WWW) at http://www.ludwig. edu.au/jpsl/jpslhome.html.     

De novo and inverse folding predictions of protein structure and dynamics

Summary In the last two years, the use of simplified models has facilitated major progress in the globular protein folding problem, viz., the prediction of the three-dimensional (3D) structure of a globular protein from its amino acid sequence. A number of groups have addressed the inverse folding problem where one examines the compatibility of a given sequence with a given (and already determined) structure. A comparison of extant inverse protein-folding algorithms is presented, and methodologies for identifying sequences likely to adopt identical folding topologies, even when they lack sequence homology, are described. Extension to produce structural templates or fingerprints from idealized structures is discussed, and for eight-membered β-barrel proteins, it is shown that idealized fingerprints constructed from simple topology diagrams can correctly identify sequences having the appropriate topology. Furthermore, this inverse folding algorithm is generalized to predict elements of supersecondary structure including β-hairpins, helical hairpins and α/β/α fragments. Then, we describe a very high coordination number lattice model that can predict the 3D structure of a number of globular proteins de novo; i.e. using just the amino acid sequence. Applications to sequences designed by DeGrado and co-workers [Biophys. J., 61 (1992) A265] predict folding intermediates, native states and relative stabilities in accord with experiment. The methodology has also been applied to the four-helix bundle designed by Richardson and co-workers [Science, 249 (1990) 884] and a redesigned monomeric version of a naturally occurring four-helix dimer, rop. Based on comparison to the rop dimer, the simulations predict conformations with rms values of 3–4 ? from native. Furthermore, the de novo algorithms can asses the stability of the folds predicted from the inverse algorithm, while the inverse folding algorithms can assess the quality of the de novo models. Thus, the synergism of the de novo and inverse folding algorthhm approaches provides a set of complementary tools that will facilitate further progress on the protein-folding problem.     

Cross-species characterisation of abundantly expressed Ochrobactrum anthropi gene products.

The identity of 45 protein spots representing 32 orthologues within the Ochrobactrum anthropi proteome within a gradient of pH 4-7, and mass range 5-90 kDa were determined across species boundaries. These proteins could be classified into 13 functional categories and establish metabolic, regulatory and translatory systems including amino acid biosynthesis, electron transport and the potential for plant symbiosis in a molecularly understudied organism. Amino acid composition and/or peptide mass fingerprinting were employed as a means to search the Swiss-Prot and OWL protein sequence databases for similarity within a broad taxonomic class of bacteria. Candidate matches from database searches could be compared and a simple multiplication matrix based on co-occurrence and rank within the top 96 most similar entries was used to provide statistical confidence. This mathematical matrix was evaluated with respect to the characterisation of O. anthropi, an unsequenced and understudied bacterium, in the light of the recent influx of DNA sequence information.     

Dimethyl isotope labeling assisted de novo peptide sequencing

Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase.     

The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints.

As a first step in establishing a proteome database for maize, we have embarked on the identification of the leaf proteins resolved on two-dimensional (2-D) gels. We detected nearly 900 spots on the gels with a pH 4-7 gradient and over 200 spots on the gels with a pH 6-11 gradient when the proteins were visualized with colloidal Coomassie blue. Peptide mass fingerprints for 300 protein spots were obtained with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer and 149 protein spots were identified using the protein databases. We also searched the pdbEST databases to identify the leaf proteins and verified 66% of the protein spots that had been identified using the protein databases. Sixty-seven additional protein spots were identified from expressed sequence tags (ESTs). Many abundant leaf proteins are present in multiple spots. Functions of over 50% of the abundant leaf proteins are either unknown or hypothetical. Our results show that EST databases in conjunction with peptide mass fingerprints can be used for identifying proteins from organisms with incomplete genome sequence information.     

Tandem mass spectrometry methods for definitive protein identification in proteomics research

Optimized procedures have been developed for the addition of sulfonic acid groups to the N-termini of low-level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two-dimensional (2-D) gel electrophoresis. The derivatized peptides were sequenced using matrix-assisted laser desorption/ionization (MALDI) post-source decay (PSD) and electrospray ionization-tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub-picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization-tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine-terminated peptides. The spectra are easily interpreted de novo, and they facilitate error-tolerant identification of proteins whose sequences have been entered into databases.     

How much peptide sequence information is contained in ion trap tandem mass spectra?

Matching peptide tandem mass spectra to their cognate amino acid sequences in databases is a key step in proteomics. It is usually performed by assigning a score to a spectrum-sequence combination. De novo sequencing or partial de novo sequencing is useful for organisms without sequenced genome or for peptides with unexpected modifications. Here we use a very large, high accuracy proteomic dataset to investigate how much peptide sequence information is present in tandem mass spectra generated in a linear ion trap (LTQ). More than 400,000 identified tandem mass spectra from a single human cancer cell line project were assigned to 26,896 distinct peptide sequences. The average absolute fragment mass accuracy is 0.102 Da. There are on average about four complementary b- and y-ions; both series are equally represented but y ions are 2- to 3-fold more intense up to mass 1000. Half of all spectra contain uninterrupted b- or y-ion series of at least six amino acids and combining b- and y-ion information yields on average seven amino acid sequences. These sequences are almost always unique in the human proteome, even without using any precursor or peptide sequence tag information. Thus, optimal de novo sequencing algorithms should be able to obtain substantial sequence information in at least half of all cases.     

Discovery and characterization of heme enzymes from unsequenced bacteria: application to microbial lignin degradation

Bacteria and other living organisms offer a potentially unlimited resource for the discovery of new chemical catalysts, but many interesting reaction phenotypes observed at the whole organism level remain difficult to elucidate down to the molecular level. A key challenge in the discovery process is the identification of discrete molecular players involved in complex biological transformations because multiple cryptic genetic components often work in concert to elicit an overall chemical phenotype. We now report a rapid pipeline for the discovery of new enzymes of interest from unsequenced bacterial hosts based on laboratory-scale methods for the de novo assembly of bacterial genome sequences using short reads. We have applied this approach to the biomass-degrading soil bacterium Amycolatopsis sp. 75iv2 ATCC 39116 (formerly Streptomyces setonii and S. griseus 75vi2) to discover and biochemically characterize two new heme proteins comprising the most abundant members of the extracellular oxidative system under lignin-reactive growth conditions.     

全新设计蛋白质的折叠机制

蛋白质全新设计和折叠研究是从两个不同的方向来理解蛋白质序列-结构-功能关系这一结构生物学重要问题. 蛋白质全新设计取得的成功实例一定程度上检验了人们对蛋白质结构和相互作用理解的准确性, 但它们中多数所表现的不同于天然蛋白质的折叠动力学特征也表明, 要达到最终的功能化实现目标还面临着不少的挑战. 本文综述了蛋白质全新设计的发展过程及现状, 蛋白质折叠研究在实验、理论及模拟方面的研究进展, 以及全新设计蛋白质的折叠机制的研究现状. 阐述了深入了解全新设计蛋白质与天然蛋白质折叠机制的不同, 可以为进一步有效地合理化设计蛋白质提供有益的参考.     

"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study

Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.     

Generating protein sequence tags by combining cone and conventional collision induced dissociation in a quadrupole time-of-flight mass spectrometer

The goal of proteomics research is to be able to identify and quantify the vast numbers of proteins within an organism or tissue. "Top-down" methods address this goal without the need for proteolytic digestion prior to mass analysis. We report here an approach for top-down protein identification that has been implemented on a commercially available, unmodified Qq-TOF mass spectrometer. Intact protein molecular ions first undergo cone fragmentation in the electrospray inlet. Conventional MS/MS is then performed on a mass selected cone fragment using CID in the Qq interface of the Qq-TOF mass spectrometer to generate a sequence tag through a pseudo-MS3 experiment. Seven proteins varying in molecular weight between 11 and 66 kDa were chosen to demonstrate applicability of method. After the molecular weight of the intact protein was determined, the cone voltage was varied to induce fragmentation. Cone fragment ions were then further dissociated using conventional CID, and the resulting MS/MS spectra were processed and analyzed for sequence tags. Sequence tags were easily identified from a MS/MS spectrum of a cone induced fragment ion both manually and through a de novo sequencing program included in the software associated with the mass spectrometer. Sequence tags were subjected to database searching using the PeptideSearch program of EMBL, and all protein sequence tags gave unambiguous search results. In all cases, sequence tags were found to originate from the n- and/or c-termini of the proteins.     

Peptic digestion of beta-casein. Time course and fate of possible bioactive peptides

Numerous peptides obtained by enzymatic digestion of food proteins have been reported to exhibit biological activities. In this study, the focus was placed on peptides of beta-casein from bovine milk after a gastro-analogous in vitro digestion with pepsin, a protease with broad specificity. In order to study the time course of the digestion, the process was stopped after specific times and the samples were subjected to HPLC separation followed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and nanoelectrospray (nanoESI) quadrupole time-of-flight (qTOF) mass spectrometry. A combined sequencing approach using de novo interpretation and databases was employed. Overall, 100% of the beta-casein sequence was covered by identifying 125 peptides of 4-84 residues in length, including 3 phosphorylated species. The results show that the peptic hydrolysis starts at the C-terminus of the protein. The release of known bioactive peptides from beta-casein following the peptic digestion under simulated gastric conditions is unlikely with a few exceptions. Furthermore, an amino acid variation was found, providing evidence for the existence of an additional genetic variant of beta-casein.     

De novo sequencing of peptides using MALDI/TOF-TOF

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.